1. Field of the Invention
The subject invention pertains to the dispensing of fluids gases or aerosols in accordance with a preselected program, or the sorting or individual particles, such as cells, of a suspension in accordance with some of their individual physio-chemical properties, such as particle size, DNA and/or protein-content of a cell. Such a method and apparatus, therefore, is useful in cytology, for cell sorting and analysis, i.e., for example, in cancer research or for the dispensing of any fluids and/or gases.
2. Description of Related Art
Two methods of practical use, and several devices based upon these methods, are known to exist for the sorting of individual particles, such as cells and chromosomes in suspension, and, using one of these methods, a droplet dispenser apparatus has also been devised recently, as is described hereinafter. The older method is often called "the jet" or "the open method"; see, for example, "Electronic Separation of Biological Cells By Volume", by M. J. Fulwyler, Science, vol. 150, pp. 910-911, Nov. 1965, and its operation is as follows:
An approximately 10 micron thick center stream, surrounded by a shealth stream of about 50-100 micron outer diameter, is ejected through an outlet of a cavity, called the nozzle chamber, to the open air in a downward direction, as the result of a pressure on the containers of the liquids of the two streams. The particles or cells are carried inside of the thin center stream to the crossing point of a very powerful laser and the optical axis of two objectives, whereby, the particle size and one of its physio-chemical components are measured through the measurement of the scattered light and the flurorescent light emitted by the particles, respectively, which, in turn, are gathered by the two objectives. The two kinds of light impulses of the passing particles or cells are converted by photomultipliers into electrical impulses, which are proportional to the two properties of the particles. From these impulses, through electronic equipment, two histograms are obtained which characterize the type of praticle of the suspension. Simutaneously, other electronic equipment analyze these impulses and determine if a particular particle is to be sorted out of the suspension in order to be collected after sorting in the corresponding container.
An ultrasonic sound wave, having a frequency of about 5.times.10.sup.4 Hz., is generated along the open stream combination by a piezoelectric transducer, so that after a few millimeters, the open stream breaks up into a stream of small droplets, source of which containing the particles to be sorted out of the suspension. During the sorting process, the stream is charged electrically at that moment when the droplets are about to break off, carrying thereby some electrical charge with them. An electrical field of a few 1000 volts/cm deflects these particles to one side or the other, depending on their electrical charge, whereafter the droplets containing the particles to be sorted are collected in their appropriate containers. Droplets containing such particles which are not to be sorted out, are not charged, and, therefore, fall vertically downward and are collected in a middle container.
Excellent cell and chromsome sortings are reported in recent works in the particle flow-through range of 700-2200 (cells or chromosomes)/sec., where, additionally, excellent distributions of the sorted pupulations are also shown; see "Analysis of Glycosaminoglycans of Flow Sorted Cells, etc." by O. C. Blair et al, Cytology, vol. 3, pp. 166-171 (b 1982), and "Separtion and Analysis of Human Chromosomes etc. ", by J. G. Collard et al, Cytometry, vol. 5, pp. 9-19 (1984). However, the rather frail shape of the open stream suggests a strong inclination towards instability, and, consequently, towards unreliability. There are, in recent works, ample comments about this type of shortcoming of the sorter; see "Multistation Multiparameter Flow Cytometry: Some influences of Instrumental Factors on System Performance", by H. M. Shapiro et al, Cytometry, vol. 4, pp. 11-19 (1983), and "Helpful Hints in Flow Cytometry and Sorting", by Ph. N. Dean, Cytometry, vol. 6, pp. 62-64 (1985).
Furthermore, in this open flow system, the stream combination can also be easily disturbed by the very same particles (cells) which are to be processed if these particles are too large relative to the stream diameter; see "The Influence of Particles on Jet Breakoff", by R. T. Stovel, J. Histochem. Cytochem., vol. 25, No. 7, pp. 813-820 (1977). Finally, the use of a very powerful light source, such as a laser, is also of some disadvantage in some cases because of its bulky nature. Such a powerful light source is needed here because only objectives of low light gathering capaabilities can be safely combined with the open jet stream in order to avoid wetting of the objective by the stream itself; and the use of high power lasers can be of some further disadvantage at some places where safety regulations also have to be taken into account.
Recently, an apparatus for the dispensing of a fluid was devised, using "the jet" method for this purpose. In this system, the fluid to be dispensed flows in a stream of uncharged droplets and the amount of its flow to the target place is controlled by another charged stream of droplets through the collision of the droplets of the two streams; see "Ballistically Controlled Nonpolar Droplet Dispensing Method and Apparatus", U.S. Pat. No. 4,341,310 to Sanjiovanni et al, issued Jul. 27, 1982. This dispenser system of "the jet" type, however, cannot be easily used for the dispensing of environmentally unfriendly fluids, and only one stream can be modulated with it.
The various shortcomings of the first method or of the systems derived from it, as described above, have been known for a long time and for this reason, the search for an improved method has been well justified.
The second method for the analysis and sorting of particles in suspension was devised by applicant around the end of 1976; see Fed. Rep. of Germany Specification P 2716095, Apr. 12, 1977, and U.S. Pat. No. 4,175,662 (1979). The second sorting method functions in the following manner: The combination of a very thin center stream and its enveloping sheath stream is generated in the nozzle chamber, in a superficially similar manner as in the first method. However, the stream combination continues to flow in the shallow main channel, itself having a cross-section comparable to that of the jet stream of the first method, and since not only the nozzle chamber and the main channel, but also the other three outlet channels, which are branching out at the end of the main channel, are covered by the same very thin, about 100 microns thick, microscope cover glass, all the various streams flow in a completely closed channel system. In this system, the cells or particles flowing inside of the center stream, are analyzed typically by an optical system, wherein the articles are illuminated by an UV-light, generated by a high pressure mercury lamp of 100 watts, giving about a 10 mwatt UV-light, through a UV-objective of very high light gathering capability, being part of a microscope of the epi-illumination type, whereby, the flurorescent light, emitted by properly dyed particles, is collected by the same objective and these light impulses are converted into electrical impulses by a photomultiplier, which is part of the same microscope system. Since the light gathering capability of this objective is around 20 to 30-fold larger than those of the first method, therefore, such a simple lamp, as described above, is able to provide the same fluorescent light as a 4 watt Argon laser, which gives out about 80 mwatts power in the quasi UV range.
After analysis, the particles continue flowing inside the center stream towards the channel branching volume, while the corresponding electrical impulses are analyzed by electronic equipment which decides if the particles are to be sorted from the suspension or not. In the "two-population" mode of sorting, where two groups of particles or cells are separted from their suspension, if the particles are to be deflected to flow in the left outlet channel, in order to be sorted out, then, through electrolysis, an amount of gas is generated in the right and middle outlet channels such taht these channels are unable to draw the electrolyte therein from the channel branching volume, and, thus, the properly arriving particles to the channel branching volume are drawn into the left outlet channel with some of the surrounding electrolyte because in this channel, gas generation did not take place and therefore its drawing capability was not reduced during the time of deflection. Naturally, if the particles are to be deflected into the right outlet channel, because they belong to the second group to be sorted out, then gas is generated in the left and middle outlet channels, while particles not belonging to either of the two group are not deflected anywhere and, therefore, they leave the flow system through the middle outlet channel. Obviously, all three partial suspensions are collected in three separate bottles, which are connected to the same suction pump, which creates about a 300 mmHg pressure, i.e., a vacuum, to draw the electrolyte through the flow system into the bottles, whereby, also larger gas bubbles are generated than at atmospherical pressure. In the "one-population" mode of opeation, the particles belonging to the group, are not deflected and, therefore, they leave the system through the middle outlet channel; all the other particles are deflected, however, intermittently into the left and right channels in order to load the sorter symmetrically, and are usually thrown away as waste. Since, in this mode of sorting, the component particles always remain in pure electrolyte, if gas is not generated in the middle outlet channel. Therefore, this mode of operation is especially useful for the sorting of live cells in accordance with some of their physio-chemical properites. Indirect tests show, however, that the "two-population" mode can also be used for the sorting of live cells, although, probably only with some limits as far as the length of the sorting operation is concerned because mouse tumor cells were found to live for as long as two hours in such electrolyte which was "contaminated" by the by-products of electrolysis of the physiological saline water.
Sorting results and additional observations have shown that devices of this method are indeed very stable and reliable. Center streams having thicknesses of about 1.0 microns were not found to waver under a 50-fold stereo microscope, where these steams were contrasted from the sheath streams by staining the former with black ink. Sorting of fluorescent latex beads of about 20 microns diameter was very successful with a device constructed on the basis of the second sorting method; this is a bead diameter where the first method or devices, constructed on the basis of it, are not usable; see the Stovel reference. The sorting of smaller beads, in the "two-population" mode, defined earlier, was also found to be very reliable, showing that this sorting method is indeed capable of sorting two groups of particles simultaneously. Similarly, the sorting of "one-population" of cells was also of high reliability. However, sorting results in the "two-population" mode have shown that very soft, jelly-like cells do not slide on the walls of the channel system when they flow with a very high velocity, because, in such a situation, they might disintegrate after such an encounter with the walls as a result of rubbing against the walls of the channel which are never perfectly smooth. However, at the end of the development of the latest sorter device, the deflection of the cells in this device was large enough to obtain excellent sporting results even in this case. At the end of these experiments, a broken electrode tip, and the form it followed, reduced sorting quality, and have shown the importance of the large enough cell deflections as was described above.
However, many of the "two-population" cell sortings were only of partial success, if the number of cells of one component was considerably larger than that of the other component. An additional observation has shown, furthermore, that, by fluorescing cells contrasted, the center stream has bent away, on the average, from that side channel where the deflecting gas bubbles were generated, favoring, thereby, further deflection of the cells. As a consequence of this, the larger cell population component was better sorted than the cells of the other population for which, in some cases, only debris was obtained as sorting results, which indicated very clearly that cells of this population were not deflected far enough from the channel walls. This shortcoming of the device has been so interpreted that the generated gas was not cleared away from the corresponding outlet channel, whereby, a reduction of the vacuum in this channel permitted a relatively stronger suction in the other outlet channel with the concommitant bending of the center stream and the rather poor sorting results of such sortings where one component population was larger than the other. The relatively slow clearing away of the generated gas in this device suggests also, rather strongly, that sortings of the order of several thousand cells per second with this system is rather unlikely.
There was no fluid dispensing attempted by the second method, for reasons of lack of interest for this type of operation at that time. However, it is easy to see that some variations of this type of operation are also readily possible with these devices if the gas bubbles are produced in accordance with a preselected program, defined by electronic equipment, such as a computer.
In U.S. Pat. No. 4,526,276 of shoor et al, the main geometrical and electrical characteristics are that the particle suspension is controlled to flow either inside of a tube, loacted quasi coaxially inside of a larger tube, or between these two tubes by using, for the flow control, the generation of an undefined type of gas production. While the patent claims contructional simplicity relative to the flow system described in U.S. Pat. No. 4,175,622, since, although the construction of the two coaxial tubes seems to cause no particular problem, on the other hand, the implementation of the control electrodes into these tubes is definitely not easier than the same type of electrode embedded into the vertical and easily observable and reachable walls of the channel system of the '622 Patent. One aspect is very clear, the two cylinder flow system permits only one-population sorting, while the channel system of the '622 Patent permits, optionally, either a single or double-population sorting, or even multiple sorting. Consequently the '276 Patent does not dominate the '622 Patent. For the sake of clarity, it is also important to note that the particles are indirectly sorted in both systems through the deflection of a small volume of the electrolyte, which surrounds the particles.